川崎病冠状动脉内皮细胞损伤的定量蛋白组学研究与生物信息学分析

Quantitative proteomics and bioinformatics analyses of human coronary artery endothelial cell injury induced by Kawasaki disease

摘要:

目的 通过同位素标记相对与绝对定量（iTRAQ）蛋白质组学技术鉴定川崎病（KD）冠状动脉内皮细胞损伤的蛋白标记物。方法 采用KD患儿血清培育的人冠状动脉内皮细胞（HCAECs）作为KD组，健康儿童血清培育的HCAECs作为健康对照组。利用iTRAQ检测两组细胞蛋白质的表达情况；采用生物信息学手段分析蛋白质数据；使用Western blot法进行蛋白标记物的验证。结果 本研究共鉴定出518个显著差异表达蛋白（差异倍数的绝对值 > 1.2且P < 0.05）。GO分析显示差异蛋白明显富集的生物过程有细胞过程、代谢过程、生物调节等；细胞组分有细胞部分、细胞、细胞器等；分子功能有结合、催化活性、分子功能调节剂等。KEGG分析显示明显富集的信号通路是核糖体、PI3K-Akt信号通路、癌症中的转录失调等。根据PPI网络结果将关系密集程度排在前9的蛋白标记物PWP2、MCM4、MCM7、MCM5、MCM3、MCM2、SLD5、HDAC2、MCM6筛选出来作为KD冠状动脉内皮细胞损伤的蛋白标记物。Western blot法验证KD组蛋白标记物HDAC2、PWP2、MCM2表达水平低于健康对照组（P < 0.05）。结论 KD患儿血清显著改变了HCAECs的蛋白表达模式并影响与心血管系统相关的信号通路，为KD的病理机制和治疗靶标提供了新的依据。

Abstract:

Objective To study the biomarkers for human coronary artery endothelial cell (HCAEC) injury induced by Kawasaki disease (KD) using isobaric tags for relative and absolute quantitation (iTRAQ) proteomics. Methods HCAECs cultured with the serum of children with KD were used as the KD group, and those cultured with the serum of healthy children was used as the healthy control group. The iTRAQ technique was used to measure the expression of proteins in two groups. The data on proteins were analyzed by bioinformatics. Western blot was used for the validation of protein markers. Results A total of 518 significantly differentially expressed proteins were identified (with an absolute value of difference fold of > 1.2, P < 0.05). The gene ontology analysis showed that the differentially expressed proteins were significantly enriched in biological processes (including cellular processes, metabolic processes, and biological regulation), cellular components (including cell parts, cells, and organelles), and molecular functions (including binding, catalytic activity, and molecular function regulators). The KEGG analysis showed that the proteins were significantly enriched in the signaling pathways of ribosomes, PI3K-Akt signaling pathway, and transcriptional dysregulation in cancer. The PPI network showed that the top 9 protein markers in relation density were PWP2, MCM4, MCM7, MCM5, MCM3, MCM2, SLD5, HDAC2, and MCM6, which were selected as the protein markers for coronary endothelial injury in KD. Western blot showed that the KD group had significantly lower expression levels of the protein markers HDAC2, PWP2, and MCM2 than the healthy control group (P < 0.05). Conclusions The serum of children with KD significantly changes the protein expression pattern of HCAECs and affects the signaling pathways associated with the cardiovascular system, which provides a new basis for the pathophysiological mechanism and therapeutic targets of KD.